Remibrutinib inhibits hives effector cells stimulated by serum from chronic urticaria patients independently of FcεR1 expression level and omalizumab clinical response

Abstract Background Despite advances in the treatment of chronic urticaria, in a significant percentage of the patients symptoms are not fully controlled with conventional approaches. New strategies under development include blocking intracellular mediators of mast cell and basophil activation. Objective We aim to investigate the effects of the Bruton's tyrosine kinase (BTK) inhibitor remibrutinib on human blood basophils and CD34+‐derived mast cells activation induced by serum obtained from chronic urticaria patients. Methods Twenty‐two patients with chronic spontaneous urticaria (mean age 52 years, 27% women) and 22 patients with chronic inducible urticaria (46 years, 27% women) were included in the study together with a sex‐matched control group. Patients were classified as responders or non‐responders to anti‐IgE therapy on the basis of their clinical data, FcεR1a expression on blood basophils and total IgE levels. Changes on CD63 expression—as an activation marker‐, were used to evaluate in vitro the response of basophils and mast cells to serum exposure and the inhibitory effects of remibrutinib. Results Remibrutinib inhibits degranulation induced by IgE cross‐linking in mast cells and basophils and also the activation triggered by factors present in the sera of spontaneous and inducible chronic urticaria patients. Patient's serum induces a greater degranulation of effector cells than controls. Activation of mast cells and basophils by patient sera and remibrutinib effects were not related to omalizumab responsiveness. Conclusion Remibrutinib inhibits activation of human basophils and mast cells induced in vitro by exposure to the serum of chronic urticaria patients independently of their response to omalizumab.


| INTRODUCTION
Urticaria is a common cutaneous and systemic condition characterized by the presence of intensely pruritic, well circumscribed, raised wheals of variable size and shape. In both chronic and acute urticaria, angioedema can be concomitantly present with hives in about a 40% of cases. Although often self-limited and benign, hives can be very uncomfortable, interfering with sleep and normal daily activity. This is especially relevant considering that in up to 30%-40% of the patients' symptoms may persist for years. In chronic urticaria (CU), lesions appear and recur over the course of 6 weeks or more. In chronic spontaneous urticaria (CSU), which accounts for the vast majority (80%) of the cases of CU, specific external triggers are absent. In contrast, in chronic inducible urticaria (CIndU), physical or environmental stimuli such as cold, heat, exercise, pressure, sunlight, or vibration are responsible for the appearance of itchy wheals and/ or angioedema. 1 The different subtypes of CU (and angioedema) have as a common pathophysiologic mechanism the release by mast cells and basophils of histamine and other proinflammatory mediators.
Degranulation may be triggered by immunoglobulin E (IgE) and/or immunoglobulin G (IgG) autoantibodies against IgE or the highaffinity IgE receptor. High-affinity receptor (FcεR1) binds the Fc region of IgE. FcεRI crosslink with IgE-autoallergen, IgE-IgG, IgG, or IgM. These mechanisms were suggested in CU, both CSU and CIndU.
It is well-known also that non-IgE and nonimmunologic mast cell activation can occur through different mechanisms. 1 In fact, many other receptors including, MRGPRX2, C5aR, PAR-1, PAR-2, CRTh2, and cytokine receptors can be activated by various signals when hives are present. 2 Antihistamines are the mainstay treatment for CU. In nonresponsive patients, biologic therapy with anti-IgE antibodies (omalizumab) is indicated. 3 Still in a significant percentage of patients it is not possible to achieve a complete control of the symptoms, highlighting the importance of exploring new therapeutic targets. 4 Bruton's tyrosine kinase (BTK) is a cytoplasmic kinase expressed in B-cells and macrophages but also in mast cells/basophils. 5 BTK is essential for signaling through FcεR1. Remibrutinib, a covalent, oral BTK inhibitor, 6 inhibited basophil activation in healthy volunteers, 7 and has shown efficacy in patients with moderate to severe CSU in a phase 2b trial. 8 There is also a growing need for biomarkers that can help to assess the severity and activity of CU and predict response to treatment. It has been previously demonstrated that the basophil baseline total IgE and FcεR1a expression can be used to characterize the response to omalizumab in CSU and CIndU patients. [9][10][11][12] Mast cells and basophils are key effector cells in the pathogenesis of CU. Using as relevant targets human hematopoietic stem cellderived mast cells and basophils from peripheral blood, we aim to investigate the effects of the BTK inhibitor remibrutinib on in vitro cell activation induced by serum obtained from CSU and CIndU patients. For CIndU, besides the UCT, specific thresholds, as defined in the guidelines, 13 were assessed at baseline and after 6 months of treatment. Treatment included second generation H1antihistamines combined with anti-IgE (omalizumab) therapy to establish the control of the disease. In total, 44 patients were included (22 with CSU and 22 with CIndU).

| Patients
Peripheral blood samples from patients with CU were analyzed before the initiation of omalizumab therapy to assess both total IgE serum levels and the FcεR1a expression on blood basophils. According to their clinical response to omalizumab, IgE and FcεR1a receptor expression on blood basophils, patients were classified as responders or non-responders to anti-IgE therapy using thresholds previously established. 9,11 CSU patients who were clinically nonresponders to omalizumab despite increasing the dose of the drug up to 600 mg, did not obtain a UAS7 <16, UCT > 12, and did not reduce their baseline UAS7 after 6 months of treatment. The dose of omalizumab was increased after the third administration due to not achieving a goal of reducing UAS7 to <16, UCT > 12, or a reduction in UAS7 after the third administration. CIndU patients nonresponders to omalizumab did not obtained UCT > 12 despite increasing the dose of the drug up to 600 mg. The dose of omalizumab was increased after the third administration if the goal of UCT>12 was not obtained.
In addition, peripheral blood samples from a control group of 22 sex-equivalent healthy adult controls (HCs) with no family or personal history of allergic asthma, allergic rhinitis, CU, or atopic dermatitis were included as reference.

| Mast cell generation
Mononuclear cells were obtained from umbilical cord blood (Banc  To assess the potential of human serum to stimulate mast cells, sensitized cells (8-10 � 10 4 cells/well) were exposed to controls' and patients' sera (10%) and incubated at 37°C. After 30 min, mast cell degranulation was stopped by placing the cells on ice for 10 min. To study the inhibitory effect of remibrutinib (LOU064, Novartis, Basel, Switzerland) on mast cells degranulation, cells were treated with 1 μM remibrutinib for 1 h at 37°C before adding the stimulus. In each experiment negative (sensitized cells without any stimulus) and positive controls (sensitized mast cells with 1 μg/ml goat anti-human IgE [Abcam, Cambridge, UK]) were included.

| Basophil stimulation
To study basophil activation, Flow CAST ® BAT was used following manufacturer's instructions. In these experiments, blood from healthy donors was incubated with or without 1 μM remibrutinib for 1 h at 37°C before the addition of controls' and patients' sera (10%).
In each experiment, a negative control consisting of unstimulated

| Statistics
Descriptive analyses were performed for each clinical value using median and range for quantitative variables and absolute (n) and

| Serum from CU patients has a greater ability to induce mast cell and basophil degranulation than sera obtained from HCs
The use of anti-IgE therapies is a major breakthrough in the management of CU. 14 It is now considered an elective choice for the treatment of antihistamine resistant CSU patients and it may be useful also in the management of some forms of CIndUs. 15 In the former case, basal IgE levels or expression of FcεR1a could anticipate the response to IgE therapies. Although there are different pathogenetic aspects of the CIndUs that remain to be defined the autoimmune mechanism has been shown, for example, for cold urticaria or cholinergic urticaria. As a next step, we examined whether serum obtained from CU patients may have the ability to activate mast cells and/or basophils. In fact, as shown in Figure 3, sera from patients with CU were capable of inducing degranulation of mast cells (upper panels) and basophils (lower panels) to a greater extent than the factors contained in the sera of healthy individuals used as controls.
When the two groups of CU patients (CSU and CIndU) were analyzed separately, at the population level and in these experimental settings sera from CIndU patients had greater power at stimulating mast cells than sera from CSU patients, whereas

| Activation of Basophils and Mast cells by patient sera is not linked to Omalizumab responsiveness
Patients who do not respond to antihistamines are presently treated with anti-IgE therapies. Although this has become a big advance in the management of these patients, still a significant number of them do not respond to IgE blockers. It has been previously shown that measurement of FcεR1 expression levels on blood basophils can help to identify those patients that will not respond to omalizumab. Next, we studied whether the sera obtained from CSU and CIndU patients which respond to omalizumab (patients with and overexpression of FcεR1a) differ from sera obtained from non-responders (patients with lower FcεR1a expression) in their ability to activate effector cells. Except for a major capability of sera from non-responder CIndU patients to activate mast cells, there were not statistically significant differences among these groups ( Figure 5). These findings support that other activating factors may be present in the serum and work independently of FcεR1, a relevant pathogenic aspect that has been suggested in previous studies. Thus in two mast cells lines expressing (LAD2) or lacking (HCM-1) the IgE receptor, a pool of sera from patients with CSU able to induce mast cells degranulation in both types of cultures and curiously most of these patients showed autologous serum skin test negative. 16 Likewise the ability to activate mast cells by low molecular weight serum fractions (100-50-30 kDa) from patients with CSU has been described. 17

| Remibrutinib inhibits in vitro basophil and mast cell degranulation induced by patient serum
The positive effect of anti-IgE therapies in the control of CU suggests a key involvement of IgE or the signaling triggered by its binding to the IgE receptors in the pathogenesis of the different types of urticaria. Remibrutinib inhibits basophil and mast cell activation induced by IgE cross-linking; we wondered whether it is also capable to block activation promoted by serum exposure. As it is seen in Figure 6A  To sum up, in the current study we describe that, in vitro, mast cell activation induced by serum from CSU and CIndU patients can be inhibited by remibrutinib. Very importantly, serum activity from both, omalizumab responders and non-responders could be equally inhibited by remibrutinib.

F I G U R E 5
Mast cells and basophils activation induced by serum factors is not related to omalizumab responsiveness. CD34 + -derived mast cells (upper panels) or blood basophils (lower panels) were incubated with serum obtained from spontaneous (CSU) or induced (CIndU) chronic urticaria patients and the upregulation of CD63 established in the relevant population by using specific monoclonal antibodies and flow cytometry. Percentages of CD63-expressing cells are shown in absolute terms (left panels) or percentage referred to the maximum activation obtained with a positive control (right panels). In these panels, patients are classified in responders or non-responders to omalizumab therapy. Mann-Whitney U test: ns no significant, *p < 0.05. Each dot in this figure represents the serum of a single individual.

F I G U R E 6
Remibrutinib inhibits effector cell activation induced by patient's serum exposure. (A), CD197 + and CD34 + -derived mast cells (upper panels) and CD193 + blood basophils (lower panels) were exposed to serum from CU patients and CD63 upregulation established by flow cytometry. Percentage of activated cells is indicated in the lower right corner of each plot. As shown in the right part of the panel, a preincubation step with remibrutinib reduces the frequency of activated cells. This is a representative experiment of more than five performed. (B) CSU or CIndU patients are classified in responders or non-responders to omalizumab therapy and the inhibitory effect of remibrutinib in each situation assessed as indicated before. Mann-Whitney U test: ns no significant. Each dot in this figure represents the serum of a single individual. (C) Representation of the effect of BTK inhibition by remibrutinib on the activation induced by patient's serum using mast cells as cellular substrate. On the left all the CU patients are represented, in the middle panel only CSU patients are shown and on the right panels only data from CIndU patients are depicted. Patients are further characterized as responsive (resp) or not (no resp) to omalizumab. Percentage of inhibition is referred to the maximum obtained with a positive control (Wilcoxon test: ****p < 0.0001).